Project Summary/Abstract The past decade has seen a resurgence of interest in understanding how the microbiota?all of the bacteria, viruses, fungi, and Archaea that normally live in and on every human being? impacts host physiology and susceptibility to disease. Although diseases in virtually every organ system have been linked with changes in the microbiota, our knowledge of whether these microbe?disease relationships are simply correlative or causal in nature is still in its infancy. There is a critical need to better understand the mechanistic basis of how commensal organisms influence disease, both from the host and bacterial perspectives. There are at least two critical bottlenecks to determining how bacteria impact host physiology: it is challenging to identify specific organisms that are causally related to disease and the lack of genetic tools for manipulation of most gram-positive commensal organisms. In earlier K08-funded research, this first research hurdle was addressed by developing an experimental approach that is able to identify microbes that are causally related to a given phenotype. Use of this method coupled with directed culture techniques led to the identification and recovery of Clostridium immunis, a previously unknown bacterial species that is able to protect colitis-prone mice from death. Having a single microbe that is clearly causally related to disease protection affords the unique opportunity to discover the bacterial factor(s) that mediates this activity. This proposal seeks to perform a comparative genomic analysis of C. immunis and several closely related bacterial species, where genomic elements will be correlated with the disease-modulating activity of each strain to identify genes that are potentially related to disease protection. Moreover, genetic tools will be developed to facilitate altering gene expression in C. immunis, thereby enabling experimental testing of the role played by the genes identified. Ultimately, these experiments will provide insight into the bacterial factors required for C. immunis-mediated protection from colitis. More broadly, the genetic tools developed will further mechanistic studies related to the microbiome while simultaneously generating the preliminary data needed to develop a competitive R01 application that further explores the host and bacterial requirements for C. immunis-mediated disease protection.